Extensive evidence suggests that two classic cell death pathways, apoptosis and necrosis, do not encompass the full variety of physiological and pathological cell death mechanisms. Our and other laboratories have established the existence of a common third pathway, termed programmed necrosis or necroptosis. Necroptosis is a regulated cell death pathway with phenotypic features of necrosis. It is activated in cells that are induced to undergo apoptosis, yet prevented from its completion. We have recently developed a potent and selective small molecule inhibitor of necroptosis, Necrostatin-1, and using this molecule have demonstrated the important role of necroptosis in various paradigms of pathologic cell death in vitro and in vivo. Discovery of necroptosis offers unique opportunity to develop novel therapies specifically targeting necrotic component of pathologic cell death, which was previously not pursued due to the notion that necrosis is an unregulated form of death. However, little is currently known regarding the specific mechanisms of activation and execution of necroptosis. Ser/Thr kinase RIP has emerged as the key upstream activator of necroptosis. Furthermore, we have recently established that RIP kinase activity is a specific cellular target of Necrostatin-1 and several other structurally unrelated potent necrostatins that were also developed in our laboratory, highlighting the critical role of RIP kinase in necroptosis. In our Preliminary studies, we developed new assays to specifically measure RIP kinase activation and necroptosis induction. We performed preliminary mass spectrometry-based characterization of RIP kinase that led to the identification of a number of novel and specific posttranslational modification (phosphorylation) events that are potentially involved in the regulation of necroptotic activity of RIP. We also demonstrated the feasibility of assessing dynamic changes in the composition of the endogenous RIP interactome using mass spectrometry analysis. Our current proposal focuses on further studies of the mechanism of necroptosis induction by RIP kinase. The specific aims of the project include confirming the role of RIP phosphorylation changes, previously identified by us, in the activation of necroptosis; characterization of RIP kinase activation process in vitro and in vivo using phospho-specific RIP antibodies and RIP kinase assay; dissection of RIP interactome using high resolution mass spectrometry followed by functional characterization of the role of RIP interacting factors in necroptosis initiation; and establishing the feasibility of the necroptosis inhibition by necrostatins as a new direction for cytoprotective therapies against acute pathologic necrosis. Overall, our studies will provide important new insights into the regulation of necroptosis through elucidating the molecular basis of the induction of the key upstream step in necroptosis, RIP kinase activation, and will validate a potential new direction for therapeutic inhibition of pathologic necrosis through selective targeting of necroptosis-specific initiation factors. Discovery of necroptosis not only provides novel insight into the basic biological regulation, but also offers new direction for therapeutic intervention against a variety of necrotizing disorders. Our proposal aims to provide further understanding of the molecular mechanism of necroptosis, which would allow us to take full advantage of this exciting discovery for development of new therapies.